National Seed Health System

ARCHIVED    Sb 3.1 – Xanthomonas axonopodis pv glycines – soybean

PROCEDURE:

1. 1. Seeds are surfaced sterilized with 95% ethyl alcohol for 30 sec.
   2. Seeds are plated on MXG selective medium amended with 1 ml of a 50 mg/ml stock solution of polymixin B sulfate/ liter.
   3. Plates are incubated at room temperature for 2 weeks.
   4. Presumptive colonies of X. campestris pv glycines are distinguished by morphological characteristics including color and size.

   Biochemical tests:

   1. Subcultures on plates of nutrient glucose agar are identified by a range of biochemical tests including oxidase reaction, acid and gas production from carbon sources, gram staining, and production of indoleacetic acid.

   Pathogencity test:

   1. Cultures are grown on nutrient glucose broth overnight and adjusted to a concentration of 10⁷ cfu/ml.
   2. Leaves of a 3-5 day old soybean plant (cultivar SJ14 or other susceptible variety) are inoculated.
   3. Plants are evaluated for symptom development after 7-10 days at 30-34^oC and 79-95% relative humidity.

RECIPES:

MXG

Prepared MXP agar

Distilled water   1000 ml

K₂HPO₄                0.8 g

KH₂HPO₄            0.6 g

Yeast extract      0.7 g

Soluble starch   0.8 g

Glucose              1.0 g

Agar                    15.0 g

Additional ingredients

1 ml methyl green (1% in 20% ETOH)=1 ml per liter of MXP, pH = 7.2-7.4 and autoclaved.

After cooling add filter sterilized:

Cycloheximide (50 mg/ml in 12.5% methanol)  1 ml

Cephalexin (50 mg/ml aqueous)  1 ml

Bacitran (10 mg/ml aqueous) 1 ml

Kasugamycin (50 mg/ml aqueous)   1 ml

REFERENCES:

Prathuangwong, S., Khandej, K., and Goto, M. 1997. Development of new methods for ecological study of soybean bacterial pustule: A semiselective medium for detecting Xanthomonas campestris pv. glycines in contaminated soybean seed. pp 197-202 in: Banpot Napompeth (ed). Proceedings of World Soybean Research Conf. V, Chiangma, Thailand 1994.